Method for detecting a polypeptide which induces interferon-γ  production

ABSTRACT

A monoclonal antibody which is specific to a polypeptide having a molecular weight of 18,500±3,000 daltons on SDS-PAGE and a pI of 4.9±1.0 on chromatofocusing. The monoclonal antibody is obtainable from hybridomas and can be used for the purification and detection of the polypeptide. The polypeptide strongly induces the IFN-γ production by immunocompetent cells with only a small amount, and dose not cause serious side effects even when administered to human in a relatively-high dose.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 09/711,899, filed Nov. 15,2000, issued as U.S. Pat. No. 6,509,449, which is a division ofapplication Ser. No. 08/558,818, filed Nov. 15, 1995, issued as U.S.Pat. No. 6,197,297, the entire contents of both applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel monoclonal antibody, moreparticularly, to a monoclonal antibody which is specific to apolypeptide capable of inducing the interferon-γ (hereinafterabbreviated as “IFN-γ”) production by immunocompetent cells.

2. Description of the Prior Art

It is known that IFN-γ is a protein which has antiviral-, antioncotic-and immunoregulatory-activities, and is produced by immunocompetentcells stimulated with antigens or mitogens. Because of these biologicalactivities, IFN-γ is expected to be used as an antitumor agent from thebeginning of the finding, and is studied energetically on clinicaltrials as a therapeutic agent for malignant tumors in general includingbrain tumors. IFN-γ preparations now commercially available are roughlyclassified into 2 groups, i.e. natural IFN-γs produced byimmunocompetent cells and recombinant IFN-γs produced by transformantsobtained by introducing into microorganisms of the species Escherichiacoli DNAs which encode such natural IFN-γs. In the above clinicaltrials, one of these IFN-γs is administered to patients as an “exogenousIFN-γ”.

Among these IFN-γs, natural IFN-γs are usually produced by culturingestablished immunocompetent cells in nutrient culture media supplementedwith IFN-γ inducers to form IFN-γs, and purifying the formed IFN-γs. Itis known that the type of IFN-γ inducers greatly influence the IFN-γyield, as well as the facility of IFN-γ purification and the safety ofthe final products. Generally, mitogens such as concanavalin A (Con A),Lens culinaris, Phytolacca americana, endotoxin and lipopolysaccharideare used as an IFN-γ inducer. However, these mitogens have problemsrelated to their molecular varieties and quality changes depending ontheir origins and purification methods, as well as the difficulty ofobtaining a desired amount of preparations with a constant IFN-γinducibility. In addition, most of these mitogens induce unfavorableside effects when administered to living bodies, and some of them evencause toxicity, so that it is substantially difficult to induce theIFN-γ production by direct administrations to living bodies.

The present inventors found in mouse liver a substance which inducesIFN-γ production during their research of cytokines produced frommammalian cells. They isolated the substance by using a variety ofpurification methods comprising column chromatography as a maintechnique, and studied the properties and features, revealing that thesubstance was a protein having the following physicochemical properties:

-   -   (1) Molecular weight Exhibiting a molecular weight of        19,000±5,000 daltons on sodium dodecyl polyacrylamide gel        electrophoresis (SDS-PAGE);    -   (2) Isoelectric point (pI) Exhibiting an isoelectric point of        4.8±1.0 on chromatofocusing;    -   (3) Partial amino acid sequence Having the partial amino acid        sequences in SEQ ID NOs:4 and 5; and    -   (4) Biological activity Inducing the IFN-γ production by        immunocompetent cells.

It can be concluded that it is a novel substance because no protein withthese physicochemical properties has been known. The present inventorscontinued studies on mouse liver cells and have found that the DNA (SEQID NO:6) of the substance consists of 471 base pairs and encodes theamino acid sequence in SEQ ID NO:7.

Based on these findings, the present inventors continued studies onhuman liver cells and have obtained a DNA which encodes another novelsubstance that induces IFN-γ production by immunocompetent cells. Theyrevealed that the substance is a polypeptide and decoded its DNA andrevealed that the polypeptide has the amino acid sequence in SEQ IDNO:1. They introduced the DNA into Escherichia coli to express thepolypeptide and obtained the polypeptide in the resultant culture in aconsiderably high yield. These findings were disclosed in JapanesePatent Application Nos.184,162/94 and 304,203/94, applied for by thepresent inventors.

As is described above, the polypeptide has a property of inducing theIFN-γ production by immunocompetent cells, and is expected to be used ina variety of fields as an IFN-γ inducer, antiviral agent, antitumoragent, antibacterial agent, immunoregulatory agent, and blood plateletenhancing agent. In general, the developments of methods for efficientlypurifying biologically active polypeptides to give a relatively-highpurity and those for assaying many samples in parallel are inevitablyrequired when the polypeptides should be incorporated intopharmaceuticals. Although the best material enabling these purificationand assay is a monoclonal antibody, none of which specific topolypeptide has been established.

SUMMARY OF THE INVENTION

In view of the foregoing, the object of the present invention is toprovide a monoclonal antibody which is specific to the polypeptide.

It is another object of the present invention to provide a hybridomacapable of producing the monoclonal antibody.

It is further object of the present invention to provide a method forpreparing the monoclonal antibody.

It is yet another object of the present invention to provide apurification method for purifying the polypeptide using the monoclonalantibody.

It is another object of the present invention to provide a detectionmethod for assaying the polypeptide using the monoclonal antibody.

The first object of the present invention is attained by a monoclonalantibody which is specific to a polypeptide having either the amino acidsequence in SEQ ID NO:1 or a homologous amino acid sequence thereunto,and induces the IFN-γ production by immunocompetent cells.

The second object of the present invention is attained by a hybridomacapable of producing the monoclonal antibody.

The third object of the present invention is attained by a process forpreparing the monoclonal antibody comprising culturing the hybridomacapable of producing the antibody in vitro, i.e. in a nutrient culturemedium, or in vivo, i.e. in an animal, and collecting the antibody fromthe resultant culture or the body fluid.

The fourth object of the present invention is attained by a purificationmethod for polypeptide comprising contacting the monoclonal antibodywith a mixture containing the polypeptide and impurities to adsorb thepolypeptide thereunto, and desorbing the polypeptide from the antibody.

The fifth object of the present invention is attained by a method fordetecting the polypeptide comprising contacting samples with themonoclonal antibody to effect immunological reaction to detect thepolypeptide.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIG. 1 is a figure of the structure of recombinant DNA pKGFHH2.

FIG. 2 is a figure of the Western blotting which shows the reactivity ofthe present purified polypeptide and human interleukin 12 with thepresent monoclonal antibody H-1mAb.

KGFHH2 cDNA: cDNA encoding the present polypeptide

Ptac: tac promoter

rrnBT1T2: terminator of ribosome RNA operon

AmpR: ampicillin resistant gene

pBR322ori: replication initiation site of Escherichia coli

DETAILED DESCRIPTION OF THE INVENTION

The monoclonal antibody according to the present invention specificallyreacts with a polypeptide having a specific amino acid sequence.

The hybridoma according to the present invention produces the monoclonalantibody when cultured in vitro.

The preparation of the monoclonal antibody according to the presentinvention facilitates its production in a desired amount.

The purification method of the polypeptide according to the presentinvention efficiently recovers it in a relatively-high quality from amixture containing the polypeptide and impurities.

In the detection method according to the present invention, only thepolypeptide in samples exhibits an immunological reaction. When theimmunoreaction level is measured by an appropriate technique, thepolypeptide can be qualitatively or quantitatively assayed.

Explaining now the present invention with reference to the Examples inthe present specification, the monoclonal antibody according to thepresent invention includes those in general which are specific to thepolypeptide having the amino acid sequence in SEQ ID NO:1 or homologousones thereunto, independently of their source, origin or class. Thehomologous amino acids include those which are obtained by replacing oneor more amino acids in SEQ ID NO:1 with other amino acids, by adding oneor more amino acids to the N- and/or C-termini in the amino acidsequence of SEQ ID NO:1, or by losing one or more amino acids in the N-and/or C-termini of the amino acid sequence in SEQ ID NO:1, whilesubstantially not losing the activity of inducing IFN-γ production byimmunocompetent cells.

The monoclonal antibody according to the present invention can beobtained by using the polypeptide or its antigenic fragments: Forexample, the antibody can be obtained by preparing hybridomas usingmammalian cells capable of infinite proliferation and antibody-producingcells collected from mammals immunized with the fragments, selectingclones of hybridomas capable of producing the monoclonal antibody, andculturing the clones in vivo or in vitro.

The polypeptide as an antigen can be obtained by culturing transformantsinto which a DNA encoding the amino acid sequence in SEQ ID NO:1 and/ora homologous one was introduced, and, generally, they are used intact orin a partially purified form. The antigenic fragments can be prepared bychemically or enzymatically hydrolyzing the wholly or partially purifiedpolypeptide, or synthesized by peptide synthesis based on the amino acidsequence in SEQ ID NO:1.

The immunization method usable in the present invention includesconventional ones: For example, antigens alone or in combination withadequate adjuvants are injected into mammals intravenously,intradermally, subcutaneously or intraperitoneally, and they are fed fora prescribed period. Any mammal can be used in the present inventionwithout special restriction as long as desired antibody-producing cellscan be obtained independently of the animal's species, weight and sex.In general, rodents such as rats, mice and hamsters are used, and fromwhich the most suitable animal is selected while evaluating thecompatibility with the above mammalian cells capable of infiniteproliferation. Depending on the species and weight of animals used, thetotal dose of the antigens is generally in the range of about 5-500 μgper animal and administered 2-5 times at an interval of 1-2 weeks. On3-5 days after the final administration, the animal's spleen isextracted and dispersed into a suspension of spleen cells as anantibody-producing cell.

The antibody-producing cells and the mammalian cells obtained in theabove are fused into a cell fusion mixture containing the objectivehybridomas. The mammalian cells capable of infinite proliferationinclude cell strains from mouse myeloma such as P3-NS1-Ag4-1 cells (ATCCTIB18), P3-X63-Ag8 cells (ATCC TIB9), SP2/O-Ag14 cells (ATCC CRL1581),and mutants thereof. The cell fusion method usable in the presentinvention includes conventional ones using an electric pulse and a cellfusion-accelerator such as polyethylene glycol and sendai virus (HVJ):For example, antibody-producing cells and such mammalian cells aresuspended in fusion media containing fusion accelerators in a ratio ofabout 1:1 to 1:10, and incubated at about 30-40° C. for about 1-5 min.Conventional media such as minimum essential medium (MEM), RPMI 1640medium, and Iscove's Modified Dulbecco's Medium (IMDM) are preferablyused as a fusion medium without addition of serums such as calf serum.

To select the objective hybridomas, the resultant cell fusion mixturewas transferred to selection media such as HAT medium, and incubated atabout 30-40° C. for about 3 days to 3 weeks to kill cells except for thehybridomas. The hybridomas were cultured in the usual manner, andantibodies secreted in the cultures were assayed for reactivity with thepolypeptide. Examples of such an assay are conventional ones fordetecting antibodies such as an enzyme immunoassay, radioimmunoassay,and bioassay. For example, “Tan-Clone-Kotai-Jikken-Manual (ExperimentalManual for Monoclonal Antibody)”, edited by Sakuji TOYAMA and TamieANDO, published by Kodansha Scientific, Ltd., Tokyo, Japan, pp. 105-152(1991) describes a variety of them. Hybridomas, which produce antibodiesthat are specific to the polypeptide, are readily cloned by limitingdilution to obtain the hybridoma according to the present invention.

The monoclonal antibody according to the present invention can beobtained by culturing the hybridoma in vivo, i.e. in animals, or invitro. For the culture conventional methods for culturing mammaliancells can be used: For example, in case of in vivo culture, themonoclonal antibody is collected from the animals' ascites and/or blood.The hybridomas H-1 and H-2 as described in the below have an enhancedproducibility of the monoclonal antibody and have a character of beingreadily cultured in vivo and in vitro. Conventional methods used topurify antibodies in general can be used to collect the monoclonalantibody from the cultures, and animal's ascites and blood. Examples ofsuch include salting out, dialysis, filtration, concentration,centrifugation, separatory sedimentation gel filtration chromatography,ion-exchange chromatography, affinity chromatography, high-performanceliquid chromatography (HPLC), gel electrophoresis, andisoelectrophoresis, and, if necessary, two or more of these techniquescan be used in combination. The resultant purified monoclonal antibodiescan be concentrated or dried into products in the form of a liquid or asolid to meet to their final use.

The present monoclonal antibody is extremely useful for purifying thepresent polypeptide on immunoaffinity chromatography. Such apurification technique comprises contacting the monoclonal antibody witha mixture containing the polypeptide and impurities such as proteinsexcept for the polypeptide to adsorb the polypeptide on the antibody,and desorbing the polypeptide from the antibody. These steps aregenerally carried out in an aqueous system. The monoclonal antibody isgenerally used in an immobilized form to gel water-insoluble carrierswhich are packed in cylindrical columns. Cultures of transformants ortheir partially purified products are fed to the columns tosubstantially adsorb the polypeptide on the monoclonal antibody. Thepolypeptide is readily desorbed from the antibody by alternating the pHaround the antibody. For example, in the case of using a monoclonalantibody of the class IgG, the adsorbed polypeptide is desorbed andeluted from the columns at an acidic pH, usually, a pH of 2-3, while inthe case of using a monoclonal antibody of the class IgM, thepolypeptide is desorbed and eluted from the columns at an alkaline pH,usually, a pH of 10-11.

The purification method according to the present invention attains arelatively-high level purification of the polypeptide with only minimumlabor cost and time. As is described above, the polypeptide has anactivity of inducing IFN-γ production by immunocompetent cells, and thepurified polypeptide can be used as an IFN-γ inducer for cell culture toproduce IFN-γ, and used in the treatment and/or the prevention of virusdiseases such as AIDS and condyloma, malignant tumors such as renalcancer, granuloma, mycosis fungoides, and cerebral tumor, and immunediseases such as articular rheumatism and allergy. If the polypeptidehas an activity of enhancing the cell cytotoxicity of killer cells, itcan be used together with interleukin 2 and/or tumor necrosis factor toimprove the therapeutic effect and reduce the side effects in thetreatment of adoptive immunity for malignant tumors including solidtumors such as lung cancer, renal cancer, and breast cancer.

The monoclonal antibody according to the present invention has arelatively-wide applicability to a variety of fields which require thedetection of the polypeptide. When used in labelled immunoassays such asradioimmunoassay, enzyme immunoassay, and fluorescent immunoassay, themonoclonal antibody can qualitatively and quantitatively detect thepolypeptide in samples instantly and accurately. In such assays, themonoclonal antibody is labelled, for example, with radioisotopes,enzymes and/or fluorescent substances prior to use. The antibodyspecifically reacts with the polypeptide to exhibit an immunoreaction,and accurately detects a slight amount of the polypeptide in samples bymeasuring the level of the immunoreaction for these labelled substances.As compared with bioassay, labelled immunoassay has the followingfeatures: It can assay many samples in parallel, reduce the assayingtime and labor cost, and provide data with a relatively high inaccuracy. Thus, the present detection method is useful for controllingthe production steps of the polypeptide and for the quality control ofthe final products. Although the present invention does not describe indetail the techniques for labelling monoclonal antibody or labellingassay because it does not in itself relate to such an invention, thesetechniques are described in detail in “Enzyme Immunoassay”, edited by P.Tijssen, translated by Eiji ISHIKAWA, published by Tokyo-Kagaku-Dojin,pp. 196-348 (1989).

The following Examples explain the present invention, and can bevariously modified by conventional methods in this art. In view of this,this invention should not be restricted to these Examples:

EXAMPLE 1

Preparation of Hybridoma H-1

Example 1-1

Preparation of Transformant KGFHH2

To a 0.5-ml reaction tube were added 8 μl of 25 mM magnesium chloride,10 μl of 10×PCR buffer, one μl of 25 mM dNTP mix, one μl of 2.5 units/μlof AmpliTaq DNA polymerase, one ng of a recombinant DNA containing thebase sequence in SEQ ID NO:2 prepared from a phage DNA clone accordingto the method in Japanese Patent Application No.304,203/94 andcontaining a DNA encoding the polypeptide in SEQ ID NO:1, and anadequate amount of a sense primer and an anti-sense primer representedby 5′-ATAGAATTCAAATGTACTTTGGCAAGCTTGAATC-3′ (SEQ ID NO:8), chemicallysynthesized based on an amino acid sequence near the N- and C-termini ofSEQ ID NO:1, and 5′-ATAAAGCTTCTAGTCTTCGTTTTGAAC-3′ (SEQ ID NO:9), andthe mixture solution was volumed up with sterilized distilled water togive a total volume of 100 μl. The mixture solution was the usual mannersuccessively incubated at 94° C. for one min, at 43° C. for one min, andat 72° C. for one min, and this sequential incubation was repeated 3times. The resultant mixture was further successively incubated at 94°C. for one min, at 60° C. for one min, and at 72° C. for one min, andthis sequential incubation was repeated 40 times to effect PCR reaction.

The resultant PCR reaction mixture and “pCR-Script SK (+)”, a plasmidvector commercialized by Stratagene Cloning Systems, California, USA,were ligated with DNA ligase to obtain a recombinant DNA which was thenintroduced with competent cell into “Escherichia coli XL-1 BlueMRF′Kan”, a microorganism commercialized by Stratagene Cloning Systems,California, USA, to transform the microorganism. The transformant thusobtained was inoculated into L-broth (pH 7.2) containing 50 μg/mlampicillin, and cultured at 37° C. for 18 hours under shakingconditions, followed by centrifuging the resultant culture to collectthe proliferated transformants, and isolating recombinant DNAs withconventional alkaline-SDS method. A part of the recombinant DNAs wasprovided, analyzed by the dideoxy method, and revealed that it containeda DNA which has cleavage sites of Eco RI and Hind III at the 5′- and3′-termini of SEQ ID NO:2, a methionine codon which initiates thepolypeptide synthesis and positions in the sites corresponding to thethose before and after the N- and C-termini of SEQ ID NO:2, and a TAGcodon which terminates the polypeptide synthesis.

The remaining recombinant DNAs were cleaved with restriction enzymes EcoRI and Hind III, and 0.1 μg of the resultant Eco RI-Hind III DNAfragment obtained with “DNA LIGATION KIT Version 2”, a DNA ligation kitcommercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, and 10 ng of“pKK223-3”, a plasmid vector commercialized by Pharmacia LKBBiotechnology AB, Uppsala, Sweden, which had been previously cleavedwith the above restriction enzymes, were ligated by incubating them at16° C. for 30 min to obtain a replicable recombinant DNA “pKGFHH2”. Byusing the competent cell method, Escherichia coli Y1090 strain (ATCC37197) was transformed with the replicable recombinant DNA pKGFHH2, andthe formed transformant “KGFHH2” was inoculated into L-broth (pH 7.2)containing 50 μg/ml ampicillin, and incubated at 37° C. for 18 hoursunder shaking conditions. The resultant culture was centrifuged tocollect the proliferated transformants, and a portion of this wastreated by the conventional SDS-alkaline method to extract therecombinant DNA pKGFHH2. As is shown in FIG. 1, the analysis of dideoxymethod revealed that, in the recombinant DNA pKGFHH2, the KGFHH2 cDNAwhich contained the base sequence in SEQ ID NO:2 was ligated to thedownstream of a Tac promoter.

Example 1-2

Production of Polypeptide from Transformant KGFHH2

An L-broth (pH 7.2) containing 50 μg/ml of ampicillin was sterilized byautoclaving, cooled to 37° C., inoculated with the transformant KGFHH2in Experiment 1-1, and incubated at the same temperature for 18 hoursunder shaking conditions to obtain a seed culture. An eighteen Lquantity of a fresh preparation of the same medium was placed in a 20-Ljar fermenter, sterilized similarly as above, cooled to 37° C.,inoculated with one v/v % of the seed culture, and cultured at the sametemperature for 8 hours under aeration and agitation conditions. Theresultant culture was centrifuged to collect cells which were thensuspended in a mixture solution (pH 7.3) consisting of 150 mM sodiumchloride, 16 mM disodium hydrogen phosphate, and 4 mM sodium dihydrogenphosphate, disrupted with ultrasound, and centrifuged to remove celldebris to obtain a supernatant.

Ammonium sulfate was added to the supernatant to give a concentration of40 w/v % and dissolved to homogeneity, and the solution was centrifugedto obtain a supernatant. The supernatant was first mixed with 150 mMphosphate buffer (pH 6.6) containing 1.5 M ammonium sulfate, then fed toa column packed with “PHENYL SEPHAROSE”, a product of Pharmacia LKBBiotechnology AB, Uppsala, Sweden, which had been previouslyequilibrated with 10 mM phosphate buffer (pH 6.6) containing 1.5 Mammonium sulfate, followed by washing the column with a freshpreparation of the same buffer, and feeding to the column a gradientbuffer of ammonium sulfate ranging from 1.5 M to 0 M in 10 mM phosphatebuffer (pH 6.6).

Fractions eluted at around 1.0 M ammonium sulfate were pooled, membranefiltered, dialyzed against 10 mM phosphate buffer (pH 6.5) at 4° C. for18 hours, and fed to a column packed with “DEAE 5PW”, a productcommercialized by Tosoh Corporation, Tokyo, Japan, which had beenpreviously equilibrated with 10 mM phosphate buffer (pH 6.5), followedby washing the column with a fresh preparation of the same buffer, andfeeding to the column a linear gradient buffer of sodium chlorideranging from 0 M to 0.2 M in 10 mM phosphate buffer (pH 6.5) whilecollecting fractions eluting at 0.05 M sodium chloride.

Thereafter, the fractions were concentrated with a membrane and fed to acolumn packed with “SUPER DEX 75”, a product of Pharmacia LKBBiotechnology AB, Uppsala, Sweden, which had been equilibrated withphosphate buffered saline (hereinafter abbreviated as “PBS”), followedby feeding to the column a fresh preparation of PBS to collect fractionscorresponding to about 18,500 daltons. Thus, an aqueous solutioncontaining about 5.2 mg of a purified protein was obtained. The totalyield throughout the purification was about 10%.

Analysis according to the method in Japanese Patent ApplicationNo.304,203/94 revealed that the purified protein had the followingphysicochemical properties: When electrophoresed in SDS-polyacrylamidegel under reducing conditions, the purified protein appeared as a mainprotein band having an IFN-γ inducibility at a position corresponding to18,500±3,000 daltons, while giving a pI of 4.9±1.0 on chromatofocusing.The amino acid sequence containing N-terminus of the purified proteinhad the amino acid sequence in SEQ ID NO:3 equal to that in SEQ ID NO:1where methionine was coupled to its N-terminus.

Example 1-3

Preparation of Hybridoma H-1

BALB/c mice, 10-week-old, were intraperitoneally injected with 20μg/mouse of a purified polypeptide, obtained by the method in Example1-2, together with a complete Freund's adjuvant. The mice were furtherinjected twice with the same dose at an interval of 2 weeks andintravenously injected with the same dose one week after the finalinjection, and their spleens were extracted and suspended to obtain acell suspension.

The spleen cells and SP2/O-Ag14 cells from mouse myeloma (ATCC CRL 1581)were suspended in RPMI 1640 medium (pH 7.2) preheated to 37° C. at celldensities of 3×10⁴ cells/ml and 1×10⁴ cells/ml, respectively, andcentrifuged to collect sediment. One ml of a serum-free RPMI 1640 medium(pH 7.2), containing 50 w/v % polyethylene glycol with an averagemolecular weight of 1,500 daltons, was added drop-wise to the sedimentover a min, and the mixture was incubated at 37° C. for a min, followedby adding drop-wise to the mixture a serum-free RPMI 1640 medium (pH7.2) up to give a total volume of 50 ml, centrifuging the mixture, andcollecting the formed sediment. The sediment thus obtained was suspendedin HAT medium, distributed to 96-well microplates in an amount of 200μl/well, and incubated at 37° C. for one week, followed by selectinghybridomas.

The amount of antibodies secreted in the supernatant in each well wasassayed on enzyme immunoassay based on the immunoreaction of theantibodies and a purified polypeptide, obtained by the method in Example1-2, and hybridomas capable of producing antibodies, which stronglyreact with the purified polypeptide, were selected. A cloned hybridomaH-1 cell capable of producing the present monoclonal antibody was in theusual manner obtained by repeatedly treating these hybridomas withlimiting dilution.

EXAMPLE 2

Preparation of Monoclonal Antibody H-1mAb and its Analysis on WesternBlot Technique

Example 2-1

Preparation of Monoclonal Antibody H-1mAb

Hybridoma H-1 cells obtained by the method in Example 1-3 were suspendedin RPMI 1640 medium (pH 7.2) supplemented with 5 v/v % calf serum togive a cell density of about 1×10⁶ cells/ml, and incubated in anincubator at 37° C. under 5 v/v % CO² conditions while scaling up theculture. When the cell density of the culture reached a prescribedlevel, 1×10⁷ cells/mouse of the proliferated hybridoma H-1 cells wereintraperitoneally injected into BALB/c mice, 8-week-old, which had beenpreviously intraperitoneally injected with 0.5 ml/mouse of pristane,followed by feeding the mice in usual manner for one week.

From the mice ascites were collected, diluted with PBS by 3 times, mixedwith ammonium sulfate to give a saturation degree of 50 w/v %, allowedto stand at 4° C. for 24 hours, and centrifuged to collect sediment. Thesediment was dialyzed against an aqueous solution of 20 mM potassiumdihydrogen phosphate (pH 6.7) at 4° C. overnight, and fed to a column ofhydroxyapatite which had been previously equilibrated with a freshpreparation of the same aqueous solution, followed by feeding to thecolumn a linear gradient potassium dihydrogen phosphate buffer (pH 6.7)ranging from 20 mM to 300 mM to obtain an aqueous solution containingthe present monoclonal antibody H-1mAb. The yield was about 5 mg permouse. Conventional analysis revealed that the antibody belongs to theclass of IgG₁.

Example 2-2

Analysis on Western Blot Technique One μg of a purified polypeptide,obtained by the method in Example 1-2, was added to a mixture solutionconsisting of 100 mg dithiothreitol, 0.5 ml of an aqueous solution of 10w/v % SDS, and one ml of glycerol, and the mixture was incubated at 37°C. for one hour and electrophoresed in SDS-polyacrylamide gel. Theresultant gel was in usual manner transferred to a nitrocellulosemembrane which was then soaked in a culture supernatant of hybridoma H-1cells for one hour, and washed with 50 mM Tris-HCl buffer (pH 7.5)containing 0.05 v/v % TWEEN 20 to remove excessive amounts ofantibodies. The membrane was further soaked for one hour in PBScontaining an anti-mouse Ig antibody. prepared from rabbits to effectimmunoreaction, washed with 50 mlvi Tris-HCl buffer (pH 7.5) containing0.05 v/v % TWEEN 20, and soaked in 50 mM Tris-HCl buffer (pH 7.5)containing 0.005 v/v % hydrogen peroxide and 0.3 mg/ml3,3′-diaminobenzidine to effect coloration.

As a control, a system using a recombinant human interleukin 12 in placeof the purified polypeptide was provided, and similarly treated asabove. Calf serum albumin (MW=67,000 daltons), ovalbumin (MW=45,000daltons), carbonic anhydrase (MW=30,000 daltons), trypsin inhibitor(MW=20,100 daltons), and α-lactalbumin (MW=14,400 daltons) were used asa marker protein. These results are shown in FIG. 2.

As is evident from FIG. 2, the monoclonal antibody H-1mAb specificallyreacted with the purified polypeptide (lane 1) obtained by the method inExample 1, but did not react with the human interleukin 12 (lane 2).This demonstrates that the present monoclonal antibody specificallyreacts with a polypeptide with a specific amino acid sequence.

EXAMPLE 3

Preparation of Hybridoma H-2 and Monoclonal Antibody H-2mAb

Hybridoma H-2, a monoclonal antibody, was similarly prepared by themethod in Example 2-1 except that P3-X63-Ag8 cells (ATCC TIB9) were usedin place of the SP/O-14Ag cells.

Example 3-2

Preparation of Monoclonal Antibody H-2mAb

The hybridoma H-2 in Example 3-1 was cultured similarly as in Example2-1, and the culture was purified to obtain about 5.6 mg of monoclonalantibody H-2mAb per BALB/c mouse. Conventional analysis revealed thatthe monoclonal antibody belongs to the class of IgM, and it specificallyreacted with a purified polypeptide obtained by the method in Example1-2 when analyzed on Western blotting technique similarly as in Example2-2.

EXAMPLE 4

Purification of Polypeptide on Immunoaffinity Chromatography

Example 4-1

Preparation of Gel for Immunoaffinity Chromatography

Eighty mg of monoclonal antibody H-1mAb, obtained by the method inExample 2-1, was weighed and dialyzed against 0.1 M borate buffer (pH8.5) containing 0.5 M sodium chloride at 4° C. overnight. Four g of“CNBr-activated Sepharose 4B”, a water-insoluble carrier commercializedby Pharmacia LKB Biotechnology AB, Uppsala, Sweden, was swollen with onemM of aqueous chloric acid solution, successively washed with a freshpreparation of the same buffer and 0.1 M borate buffer (pH 8.5)containing 0.5 M sodium chloride, admixed with an about 10 ml of theaqueous monoclonal antibody solution obtained in the above, andsuccessively incubated at ambient temperature and at 4° C. overnightunder gentle stirring conditions. Thereafter, the resultant gel wassuccessively washed with one M aqueous ethanol amine solution (pH 8.0),0.1 M borate buffer (pH 8.5) containing 0.5 M sodium chloride, and 0.1 Macetate buffer (pH 4.0), and these washing steps were repeated 5 times.Finally, the gel was washed with PBS to obtain a gel for immunoaffinitychromatography. Conventional analysis revealed that about 6 mgmonoclonal antibody H-1mAb linked to one ml of the gel.

Example 4-2

Purification of Polypeptide by Immunoaffinity Chromatography

Ten ml of the gel for immunoaffinity chromatography in Example 4-1 waspacked in a plastic cylindrical column, washed with PBS, and fed with 10ml of a Phenyl Sepharose eluted fraction containing about 0.1 mg/ml ofthe polypeptide obtained by the method in Example 1-2. The column waswashed with a fresh preparation of PBS, and fed with 0.1 M glycine-HClbuffer (pH 2.5) containing one M sodium chloride to collect fractionswith an IFN-γ inducing activity. The fractions were pooled, dialyzedagainst PBS at 4° C. overnight, concentrated and assayed for the IFN-γinducing activity and the protein content, revealing that thispurification procedure yielded a purified polypeptide with a purity of95 w/w % or higher in a yield of about 100%.

EXAMPLE 5

Detection of Polypeptide on Enzyme Immunoassay

Rabbits were in the usual manner immunized with a purified polypeptideobtained by the method in Example 1-2, and their blood collected.Immunoglobulin G antibody was isolated from the blood, dissolved in PBSto give a concentration of 20 μg/ml, and the solution was distributedinto 96-well microplates in an amount of 100 μl/well. The microplateswere incubated at ambient temperature for 3 hours, followed by removingsolutions containing IgG from the microplates, adding PBS containing onew/v % calf serum albumin to the microplates in an amount of 200 μl/well,and allowing them to stand at 4° C. overnight.

Phosphate buffered saline was removed from the microplates which werethen washed with PBS containing 0.05 v/v % TWEEN 20, and injected with100 μl/well of a solution prepared by appropriately diluting a purifiedpolypeptide, obtained by the method in Example 1-2, with PBS containing0.5 w/v % calf serum albumin, followed by reacting the mixture solutionat ambient temperature for 2 hours under shaking conditions. Themicroplates were washed with PBS containing 0.05 v/v % TWEEN 20, andinjected with 100 μl/well of a solution containing a monoclonal antibodyH-1mAb labelled with biotin, followed by reacting the mixture solutionat ambient temperature for 2 hours under shaking conditions, washing themicroplates with PBS containing 0.05 v/v % TWEEN 20, injecting with 100μl/well of a solution containing a complex of horseradish peroxidase andstreptoavidin, and further reacting the resultant mixture at ambienttemperature for 2 hours under shaking conditions. Then, the microplateswere washed with PBS containing 0.05 v/v % TWEEN 20, and the activity ofthe horseradish peroxidase linked to the purified polypeptide wasmeasured for absorbance at a wavelength of 492 nm usingo-phenylenediamine as a substrate. The results are shown in Table 1.

TABLE 1 Concentration of Absorbance Relative error polypeptide (pg/ml)at 492 nm* (%) 1,000 1.51 ± 0.05 3.3 500 0.93 ± 0.05 5.4 250 0.55 ± 0.035.5 100 0.25 ± 0.02 8.0 50 0.137 ± 0.007 5.1 25 0.080 ± 0.007 8.8 00.024 ± 0.007 — Note: The symbol “*” means a statistical value oftriplet.

As is evident from the results in Table 1, the detection methodaccording to the present invention accurately assays the polypeptide inthe range of about 50-1,000 pg/ml.

EXAMPLE 6

Detection of Polypeptide on Radioimmunoassay

Rabbits immunized were in the usual manner with a purified polypeptideobtained by the method in Example 1-2, and their blood was collected,followed by isolating IgG antibody. The antibody was absorbed in theusual manner on polystyrene beads for radioimmunoassay, and allowed tostand in PBS containing 2 w/v % calf serum albumin at 4° C. overnight toobtain an immobilized antibody.

One bead was placed in a test tube, soaked in 0.2 ml of a solutionprepared by diluting a purified polypeptide, obtained by the method inExample 1-2, with PBS containing 0.5 w/v % calf serum albumin, andallowed to stand at 4° C. for 4 hours. Then, the bead was washed withPBS containing 0.05 v/v % TWEEN 20 and 0.5 w/v % calf serum albumin,soaked in 0.2 ml (1×10⁵ cpm) of a solution containing a monoclonalantibody H-2mAb, obtained by the method in Example 3-2 and labelled with¹²⁵I, and allowed to stand at 4° C. overnight. After removing anexcessive amount of ¹²⁵I-labelled antibody, the bead was washed with PBScontaining 0.05 v/v % TWEEN 20 and 0.5 w/v % calf serum albumin,followed by counting the radioactivity of the bead on a gamma-counter.The results were in Table 2.

TABLE 2 Concentration of Count* Relative error polypeptide (pg/ml) (cpm)(%) 1,000.0  6,900 ± 200 2.9 500.0 4,100 ± 20 0.5 250.0 2,390 ± 50 2.1125.0 1,590 ± 70 4.4 62.5   880 ± 10 1.1 0   700 ± 20 — Note: The symbol“*” means a statistical value of triplet.

As is evident from the results in Table 2, the present detection methodaccurately assays the polypeptide in the range of about 100-1,000 pg/ml.

As is described above, the present monoclonal antibody specificallyreacts with a polypeptide which induces IFN-γ production byimmunocompetent cells. Therefore, the monoclonal antibody is widely usedin the purification and detection of the polypeptide, and is prepared ina desired amount by a preparation using hybridomas.

The present invention with these significant functions and effects is asignificant invention which greatly contributes to this field.

While there has been described what is at present considered to be thepreferred embodiments of the invention, it will be understood thatvarious modifications may be made therein, and it is intended to coverin the appended claims all such modifications as fall within the truespirit and scope of the invention.

1. A method for quantitatively or qualitatively detecting a polypeptidein a sample, comprising: (a) contacting said sample with a monoclonalantibody specific for said polypeptide to adsorb said polypeptide onsaid monoclonal antibody; and (b) measuring the level of immunoreactionbetween said polypeptide and said monoclonal antibody to determine thepresence or amount of said polypeptide in said sample by usingradioimmunoassay, enzyme immunoassay, or fluorescent immunoassay,wherein said polypeptide is capable of inducing interferon-γ productionin immunocompetent cells and has the amino acid sequence of SEQ ID NO:1,where the symbol Xaa represents isoleucine or threonine, or has an aminoacid sequence which is obtained by replacing one or two amino acids inSEQ ID NO:1 with other amino acids, by adding one or two amino acids tothe N- and/or C-termini in the amino acid sequence of SEQ ID NO:1,and/or by deleting one or two amino acids from the N- and/or C-terminiof the amino acid sequence of SEQ ID NO:1, while substantially notlosing the activity of inducing IFN-γ production by immunocompetentcells.